1. Field of the Invention
The present invention relates to formation of a stable hybrid between a sample nucleic acid and a probe immobilized on a substrate. The present invention also relates to a probe for use in forming such a hybrid, a PCR primer, a probe carrier and designing methods thereof.
2. Related Background Art
As is represented by the Human Genome Project, gene sequences of various types of organisms have been elucidated and the relationships between genes and the mechanism of life activity, diseases, constitutional predispositions and so forth are investigated. It is now understood that by determining the presence or absence of a certain gene or its (expression) amount, not only the feature or type of a disease but also the effective therapy thereof may be determined.
Various methods have been proposed for detecting the presence or absence of a specific gene in a test sample and determining the expression amount of the gene, among which, the following method is applicable to a wide variety of samples. This method comprises the steps of selecting (determining) a partial nucleotide sequence specific to a target gene, and detecting the presence or absence or the expression amount of the partial sequence, thereby determining the presence or absence of the gene or its expression amount. More specifically, according to this method, a nucleic acid (probe) of which sequence is complementary to the selected partial sequence is prepared, and the probe and a test sample are hybridized to detect whether a hybrid is formed or not by certain means, whereby the presence or absence of the gene in the sample is determined.
This method of detecting a specific nucleic acid by using hybridization can be used in both solid and liquid phases. For example, hybridization in a liquid phase is performed as follows: a probe labeled with a labeling agent of which spectroscopic property changes when the probe becomes double stranded; then a test sample is added to a solution of the probe; and spectroscopic change is measured. In this way, the presence or absence of a specific nucleic acid can be determined.
On the other hand, hybridization on a solid is typically performed as follows: a probe is immobilized onto a substrate; then, a sample labeled with a detectable labeling agent is applied to the substrate; and a signal emitted from the labeling agent on the substrate is measured. Typical examples are microarrays where probes are immobilized on a flat glass or metal substrate, and beads that are fine particles having immobilized probes thereon. Solid-phase hybridization is preferably used for the following reasons: B/F separation (separation of bound form and free form) is easy; detection can be performed in a minute area and with high sensitivity; if a plurality of types of probes are discretely arranged, simultaneous detection of a plurality of items can be made; and solid substrates are easy to handle or use.
For example, U.S. Pat. No. 6,410,229 to Affymetrix Inc. (USA) discloses a method for detection and quantification of a specific nucleic acid in a sample by hybridizing a labeled nucleic acid with an oligo DNA synthesized on a flat substrate, and detecting fluorescence emitted from the hybrid. On the other hand, Japanese Patent Publication No. 2001-128683 (Fuji Photo Film Co., Ltd.) discloses detection of 22 mer ssDNA by using a DNA array prepared using a substrate to which amino groups had been introduced beforehand.